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- PDB-3a2k: Crystal structure of TilS complexed with tRNA -

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Basic information

Entry
Database: PDB / ID: 3a2k
TitleCrystal structure of TilS complexed with tRNA
Components
  • bacterial tRNA
  • tRNA(Ile)-lysidine synthase
KeywordsLIGASE/RNA / Ligase / RNA / pseudo-knot / LIGASE-RNA COMPLEX
Function / homology
Function and homology information


tRNAIle-lysidine synthase / ligase activity, forming carbon-nitrogen bonds / tRNA modification / ATP binding / cytoplasm
Similarity search - Function
tRNA (Ile)-lysidine synthase / Uridine Diphospho-n-acetylenolpyruvylglucosamine Reductase; domain 3 - #60 / Lysidine-tRNA(Ile) synthetase, C-terminal / tRNA(Ile)-lysidine synthase , substrate-binding domain / TilS substrate binding domain / TilS substrate C-terminal domain / TilS substrate C-terminal domain / tRNA(Ile)-lysidine synthase / tRNA(Ile)-lysidine synthase, N-terminal / tRNA(Ile)-lysidine/2-thiocytidine synthase, N-terminal ...tRNA (Ile)-lysidine synthase / Uridine Diphospho-n-acetylenolpyruvylglucosamine Reductase; domain 3 - #60 / Lysidine-tRNA(Ile) synthetase, C-terminal / tRNA(Ile)-lysidine synthase , substrate-binding domain / TilS substrate binding domain / TilS substrate C-terminal domain / TilS substrate C-terminal domain / tRNA(Ile)-lysidine synthase / tRNA(Ile)-lysidine synthase, N-terminal / tRNA(Ile)-lysidine/2-thiocytidine synthase, N-terminal / PP-loop family / Uridine Diphospho-n-acetylenolpyruvylglucosamine Reductase; domain 3 / HUPs / Rossmann-like alpha/beta/alpha sandwich fold / Arc Repressor Mutant, subunit A / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / tRNA(Ile)-lysidine synthase
Similarity search - Component
Biological speciesGeobacillus kaustophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 3.65 Å
AuthorsNakanishi, K. / Bonnefond, L. / Ishitani, R. / Nureki, O.
CitationJournal: Nature / Year: 2009
Title: Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase.
Authors: Nakanishi, K. / Bonnefond, L. / Kimura, S. / Suzuki, T. / Ishitani, R. / Nureki, O.
History
DepositionMay 23, 2009Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 20, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jan 24, 2018Group: Experimental preparation / Category: exptl_crystal_grow
Item: _exptl_crystal_grow.pdbx_details / _exptl_crystal_grow.temp

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: tRNA(Ile)-lysidine synthase
B: tRNA(Ile)-lysidine synthase
C: bacterial tRNA
D: bacterial tRNA


Theoretical massNumber of molelcules
Total (without water)155,8164
Polymers155,8164
Non-polymers00
Water0
1
A: tRNA(Ile)-lysidine synthase
C: bacterial tRNA


Theoretical massNumber of molelcules
Total (without water)77,9082
Polymers77,9082
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6130 Å2
ΔGint-58 kcal/mol
Surface area33500 Å2
MethodPISA
2
B: tRNA(Ile)-lysidine synthase
D: bacterial tRNA


Theoretical massNumber of molelcules
Total (without water)77,9082
Polymers77,9082
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5880 Å2
ΔGint-47 kcal/mol
Surface area33320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)85.734, 157.396, 208.177
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
12
22

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection details
111chain A
211chain B
112chain C
212chain D

NCS ensembles :
ID
1
2

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Components

#1: Protein tRNA(Ile)-lysidine synthase / tRNA(Ile)-lysidine synthetase / tRNA(Ile)-2-lysyl-cytidine synthase


Mass: 53188.430 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus kaustophilus (bacteria) / Strain: HTA426 / Gene: GK0060, tilS / Plasmid: pET28c / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) codonplus
References: UniProt: Q5L3T3, Ligases; Forming carbon-nitrogen bonds; Other carbon-nitrogen ligases
#2: RNA chain bacterial tRNA


Mass: 24719.684 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: RNA WAS PREPARED BY IN VITRO TRANSCRIPT

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

Crystal
IDDensity Matthews (Å3/Da)Density % sol (%)
14.572.7
2
Crystal grow
Temperature (K)Crystal-IDMethodpHDetails
2931vapor diffusion, hanging drop6.545mM Na-cacodylate (pH 6.5), 200mM NaCl, 225mM ammonium acetate, 11mM CaCl2, 7.2 % PEG 4000, VAPOR DIFFUSION, HANGING DROP, temperature 293K
2932vapor diffusion, hanging drop5.550mM Na-cacodylate buffer (pH 5.5), 280mM ammonium acetate, 12mM CaCl2, 4% (w/v) PEG3350, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21001
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONPhoton Factory AR-NW12A11
SYNCHROTRONSPring-8 BL41XU20.97917, 0.97942, 0.99500
Detector
TypeIDDetectorDate
ADSC QUANTUM 2101CCDJun 13, 2007
ADSC QUANTUM 3152CCDOct 3, 2007
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2MADMx-ray1
Radiation wavelength
IDWavelength (Å)Relative weight
111
20.979171
30.979421
40.9951
ReflectionResolution: 3.65→50 Å / Num. all: 31287 / Num. obs: 31287 / % possible obs: 96.6 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 4.9 % / Biso Wilson estimate: 112.13 Å2 / Rsym value: 0.06 / Net I/σ(I): 18.2
Reflection shellResolution: 3.65→3.71 Å / Redundancy: 2.4 % / Rmerge(I) obs: 0.22 / Mean I/σ(I) obs: 2 / Num. unique all: 1284 / Rsym value: 0.39 / % possible all: 81.1

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Processing

Software
NameVersionClassification
PHENIX(phenix.refine)refinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
SHARPphasing
RefinementMethod to determine structure: MAD / Resolution: 3.65→43.751 Å / SU ML: 2.26 / σ(F): 0.28 / Phase error: 26.46 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2664 1553 5 %RANDOM
Rwork0.2158 ---
all0.2183 31067 --
obs0.2183 31067 96.85 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 56.388 Å2 / ksol: 0.228 e/Å3
Displacement parametersBiso mean: 149.09 Å2
Baniso -1Baniso -2Baniso -3
1-12.4002 Å20 Å20 Å2
2---9.8613 Å20 Å2
3----2.5388 Å2
Refinement stepCycle: LAST / Resolution: 3.65→43.751 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7448 3276 0 0 10724
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00911280
X-RAY DIFFRACTIONf_angle_d1.6515972
X-RAY DIFFRACTIONf_dihedral_angle_d27.1365046
X-RAY DIFFRACTIONf_chiral_restr0.0911854
X-RAY DIFFRACTIONf_plane_restr0.0061504
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A3724X-RAY DIFFRACTIONPOSITIONAL
12B3724X-RAY DIFFRACTIONPOSITIONAL0.046
21C1638X-RAY DIFFRACTIONPOSITIONAL
22D1638X-RAY DIFFRACTIONPOSITIONAL0.045
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.65-3.76780.42731210.392232686
3.7678-3.90230.33881380.3449259495
3.9023-4.05850.35771370.3163261797
4.0585-4.2430.31261400.2585265497
4.243-4.46650.29791410.2195268398
4.4665-4.74610.2411420.1828270898
4.7461-5.1120.23731440.1704272799
5.112-5.62550.24141440.1655274199
5.6255-6.43730.23451450.1611276099
6.4373-8.10190.22091480.1422799100
8.1019-43.75460.21911530.197290598
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.61671.32070.82061.5541-1.9670.9265-0.131-0.32141.11610.15940.00750.1527-0.2428-0.34240.00040.74820.2567-0.01781.1605-0.41551.2106-58.942911.014620.0632
24.22010.54160.27893.70081.8012.9766-0.4914-0.2286-0.120.7007-0.25681.70760.2576-0.3934-1.04790.60360.14990.00271.0669-0.04990.395-38.2958-16.848130.8823
32.45790.9346-2.09581.5611-3.27590.7949-0.37850.26370.57810.74170.02990.3592-0.2426-0.3831-0.03261.1836-0.08690.29470.701-0.2030.8359-28.6949-47.896347.8177
43.47910.6004-0.1150.36220.35744.1572-0.07970.00690.2709-0.1470.0768-0.3958-0.00420.5240.00010.6476-0.1039-0.0110.9866-0.1771.4251-16.992811.293319.8886
54.8722.1521.85196.933-1.4252.2981-0.35810.40970.215-0.61260.8248-1.7739-0.70190.6929-0.23940.48020.15630.18970.71670.10130.5806-37.4088-1.769-7.1704
63.79741.0448-0.59671.3382-0.5012.87-0.3892-0.26660.2992-0.57860.0775-0.35920.3011-0.078-0.00041.06550.02050.19930.4732-0.06310.9055-46.6649-13.1301-40.7541
71.10742.02120.21532.045-0.43150.10850.1206-0.9881-0.0077-0.401-0.3634-0.9503-0.5199-0.4692-01.4154-0.1023-0.2171.0582-0.42661.2404-11.5987-24.810549.3807
80.9397-0.23191.86151.0547-0.9055-0.66270.7559-0.85581.33811.1285-0.5248-0.5956-0.6255-0.65750.00021.6389-0.0775-0.23221.7837-0.21491.2896-20.5759-6.09949.4726
9-0.0248-1.69750.01190.8642-2.777-0.7154-0.1618-0.3012-0.52820.75580.6420.7012-0.5105-0.5331-0.00011.39460.3782-0.02641.7475-0.22371.4337-43.58583.031439.6203
102.3009-0.60990.88011.67611.06183.8562-0.02390.40161.25120.4669-1.26450.2794-0.5319-0.6839-0.00141.36170.5347-0.02461.1907-0.11891.8295-63.42745.7271-27.1457
110.2947-0.7711.73240.81412.94010.4492-0.4538-0.80551.49380.2511-0.54050.9453-1.5253-1.0751-0.00062.28230.561-0.04391.7161-0.00781.9895-54.524119.4183-14.3359
120.17740.2989-0.8314-1.43631.16261.89930.0768-0.21671.10920.7263-0.45910.6229-1.90351.0396-01.8977-0.2946-0.27271.6750.01841.7644-32.075618.88690.0111
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain A and resid 1:236
2X-RAY DIFFRACTION2chain A and resid 237:332
3X-RAY DIFFRACTION3chain A and resid 333:462
4X-RAY DIFFRACTION4chain B and resid 1:236
5X-RAY DIFFRACTION5chain B and resid 237:332
6X-RAY DIFFRACTION6chain B and resid 333:462
7X-RAY DIFFRACTION7chain C and (resid 1:7 or resid 50:99)
8X-RAY DIFFRACTION8chain C and (resid 8:26 or resid 46:49)
9X-RAY DIFFRACTION9chain C and (resid 27:45)
10X-RAY DIFFRACTION10chain D and (resid 1:7 or resid 50:99)
11X-RAY DIFFRACTION11chain D and (resid 8:26 or resid 46:49)
12X-RAY DIFFRACTION12chain D and (resid 27:45)

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